IN SITU detection of early stages and late stages HPV infection

ABSTRACT

Embodiments of the invention provide methods, polyclonal antibodies, monoclonal antibodies, assays, and kits for detecting HPV infection, including infection by various HPV genotypes, early and/or late HPV-associated or HPV-specific proteins or antibodies. Mononoclonal antibodies are used to detect oncogenic high risk and low risk HPV types in a single assay, which is not limited to assay type or format. Useful tools for specific detection of invasive cervical cancer are provided. Cervical cancer biomarkers are identified and can be used in a detection method for early stage precancerous lesions as well as late stage cancer progression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent application Ser. No. 61/131,991, filed Jun. 13, 2008, and U.S. provisional patent application Ser. No. 61/192,912, filed Sep. 22, 2008. Each of the aforementioned related patent applications is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Infection by human papillomaviruses (HPV) at specific epithelium cells to induce epithelial proliferations plays an important role for cervical carcinogenesis. About 99 percent of confirmed cervical cancer cases are found to be associated with HPV infection with biopsy-confirmed squamous intraepithelial lesions (SIL) or cervical intraepithelial neoplasia (CIN). The incidence of HPV infection, primarily transmitted through sexual contact, is highest among young women and about 20 millions of sexually active men and women worldwide are currently infected. Approximately 1% of the population has genital warts and 4% of women have cervical precancerous lesions, such as low grade of squamous intraepithelial lesion (LSIL) or high grade of squamous Intraepithelial lesion (HSIL) or atypical squamous cells of undetermined significance (ASCUS).

The presence of these lesions, preferentially observed in women aged 35-40 yrs, are at high risk of progression toward invasive cervical cancer. It is general thought that persistent infection of human papillomavirus (HPV) is essential for developing precancerous epitheliual lesions. Infection of high-risk types HPV for women with LSIL may or may not progress to HSIL. In fact, remission occurs in majority of LSIL human subjects while some progress to HSIL. Although 99.7% of cervical cancers are HPV positive, integration of viral genome into the host genome is required to facilitate the necessary genes to express for developing into HSIL or cancer. In fact, only one in every 10 women with persistent HPV infection may develop into higher grades of CIN lesions, such as cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2, and CIN3, respectively), and a portion of these epitheliual lesion cases may ultimately progress into cervical cancer.

In the past, screening for cervical cancer is based on conventional cytology screening tests, e.g., obtaining papanicolaou (Pap) smears for cytological staining tests, and suspicious smears are followed up with colposcopy, and/or histological biopsy. The use of these cytological screening tests contributes to a reduction in the mortality of cervical cancer. However, due to subjective test criteria, there are various drawbacks for pap smear tests: difficulty in obtaining samples, poor inter- and intra-observer agreement, high rates of false negatives nd false positives, requiring specialized labs staffed with highly trained personnel, and inability to identify the majority of HPV-infected human subjects. More reproducible assays are needed to improve the current screening tests to avoid unnecessary medical intervention and psychological distress for the affected women. The current conventional cervical cytology screening tests have sensitivity varied from about 30% to about 87%.

Detecting HPV infection by nucleic acid tests, such as “DNA Hybrid Capture”, has been developed with high assay sensitivity, but are still not ideal, due to not only its high cost, assay operation procedures, the requirements for facility, equipment, and highly trained personnel, but also its very low positive predictive value (PPV) in cervical intraepithelial neoplasia (CIN) testing samples. Assay like PreTect HPV-Proofer® provides the detection of E6/E7 mRNA with sensitivity equivalent to HPV Hybrid Capture tests with higher positive predictive value; but cannot directly detect E6/E7 oncoproteins in situ. In addition, DNA testing could not differentiate disease stages after HPV infection nor the diagnosis of different cell lesions (e.g., cannot differentiate LSIL from HSIL, nor CIN lesions from non-transforming latent or remissive viral infection). What is needed is a low cost, simple, sensitive and specific assay that can be performed on routine practice of a clinical lab or doctor office and capable of detecting early stages of epithelial lesions, distinguish LSIL from HSIL, or predicting the risk of progression into cervical cancer.

Known protocols for producing monoclonal antibodies are generally unsuitable for the production of anti-HPV monoclonal antibodies and cannot be used in immunocytochemical diagnostic tests performed on human subjects of general population. This is because antibodies produced by these protocols will not necessarily react with the naturally occurring HPV viral proteins in infected human cells. It is thought that the epitopes recognized by antibodies if generated by conventional protocols will not necessarily be those epitopes which are resistant to the harsh procedures involved in standard sampling, fixing and storing of clinical specimens. In addition, three problems exist in clinical HPV detection. One is that HPV proteins in clinical samples are present in very small quantities. Secondly, there are too many HPV types and most HPV types present in clinical samples are not known or systemically identified due to the lack of available antibodies. Third, HPV virus can not be cultured in labs by standard tissue culture techniques. Thus, there is no available HPV proteins purified to large quantities as immunogens for generating anti-HPV antibodies, and there is no available HPV proteins or purified anti-HPV antibodies to recognize anti-viral antibodies or viral proteins present in clinical samples for clinical HPV detection.

Infections by only about 15 HPV types (out of more than 100 available HPV types) are at high risk of developing into cervical intraepithelial neoplasia (CIN) or cervical cancer. Among them, around 70% of reported cervical cancer cases and 50% of reported CIN 2 and CIN 3 cases are caused by two high risk HPV types, i.e., HPV type-16 and HPV type-18. However, some progressive cervical cancer cases are reported to be infected by low risk HPV types, while infection of some high risk HPV types will never progress into cervical cancer. Infections by these two prevailing high risk HPV types do not correlate with tumor development or cancer progression. It seems important to identify those HPV-infected human subjects that express particular oncogenic proteins rather than just identify HPV infection by high risk types.

Thus, there is a need for detect the expression of HPV-related oncoproteins in clinical samples as these oncoproteins may better serve as cervical cancer biomarkers to better predict the risk in developing into high grade of cell lesions or cervical cancer-related diseases. There is also a need to develop anti-HPV antibodies and appropriate HPV immunoassays to detect the presence of invasive cervical cancer and/or HPV-related oncoproteins as cervical cancer biomarkers and predict the risk for malignant transformation of epithelial lesions into cervical cancer.

SUMMARY OF THE INVENTION

Embodiments of the invention provide various immunoassays for in situ detection of HPV proteins using various monoclonal antibodies against recombinant HPV proteins such that infection by high risk and/or low risk HPV types can be detected by a single specific monoclonal antibody and/or a general pan antibody. In one embodiment, a method of detecting papillomavirus infection in a human subject includes conducting an immunological assay on a clinical sample of the human subject; and staining of a nuclear portion of a human cell from the clinical sample using one or more monoclonal antibodies capable of binding to HPV early viral proteins. In another embodiment, a method of detecting papillomavirus infection in a human subject include conducting an immunohistochemistry assay on humans cells from a clinical sample of the human subject using one or more antibodies generated against one or more purified recombinant papillomavirus proteins and comparing the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cell from the clinical sample. Another method of detecting papillomavirus infection in a human subject includes conducting an immunohistochemistry assay on a clinical sample of the human subject using two or more antibodies capable of binding to a HPV viral protein selected form the group consisting of E6, E7, L1 proteins, and combinations thereof, and comparing the staining of the human cell by the two or more antibodies, wherein positive staining of the human cell by at least one of the two or more antibodies indicates papillomavirus infection in the human subject.

In one aspect, the invention provides an anti-HPV monoclonal antibody capable of being used in an immunological assay to stain a cytoplasmic portion of a HPV-infected human cell from a human subject with a late disease stage. In another aspect, the invention provides an anti-HPV E7 monoclonal antibody and anti-HPV E6 monoclonal antibody capable of being used in an immunological assay to stain a nuclear portion of a human cell from a human subject to indicate HPV infection at a disease stage. The staining of the nuclear portion of the HPV infected human cell indicates early stage HPV infection, whereas the staining of the cytoplasmic portion of the HPV infected human cell indicates dysplasia progression to a late disease stage by HPV infection. The anti-HPV monoclonal antibody used in the immunological assay may be an anti-HPV E7 monoclonal antibody, anti-HPV E6 monoclonal antibody, anti-HPV L1 monoclonal antibody, and combinations thereof. In another aspect, the invention provides an anti-HPV E7 monoclonal antibody and HPV E7 protein as a biomarker used for detection of cervical cancer progression.

In addition, the invention provides methods of performing one or more immunological assays, including immunohistochemistry assays and immunocytological assays. Monoclonal antibodies highly specific for HPV viral proteins are also provided to be used in the HPV-detecting immnological assays. In one embodiment, a nuclear portion of an epithelial tissue sample is stained with the anti-HPV monoclonal antibody to indicate HPV infection in general. In another embodiment, a cytoplasmic portion of an epithelial tissue sample is stained with the anti-HPV monoclonal antibody to indicate dysplasia progression by HPV infection.

In still another aspect, a kit for performing an immunological assays on a clinical sample is provided and includes an anti-HPV monoclonal antibody capable of staining a nuclear portion of a HPV infected human cell from a human subject to indicate HPV infection and capable of staining a cytoplasmic portion of the HPV infected human cell to indicate HPV infection at a late disease stage. In one aspect, a kit for detecting papillomavirus infection in a human subject may include an anti-HPV monoclonal antibody for performing an immunological assays on a clinical sample of the human subject, capable of staining a nuclear portion of one or more human cells from the clinical sample to compare the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cells.

SUMMARY OF DRAWINGS

FIG. 1A shows the representative image of the squamocarcinoma (SCC) tissue from tissue microarray stained by IHC using an anti-E7 monoclonal antibody.

FIG. 1B shows the representative image of the normal epithelium (15 mm away from the tumor tissue) of the SCC subject from FIG. 1A.

FIG. 1C shows the representative image of another SCC sample from tissue microarray stained by IHC using the same anti-E7 monoclonal antibody.

FIG. 1D shows the magnified representative image of the tumor cells stained in cytoplasm from FIG. 1C.

FIG. 2A shows the representative image of the tumor cells of adenocarcinoma (ADC) sample stained by IHC using an anti-E7 monoclonal antibody.

FIG. 2B shows the representative image of the corresponding normal epithelium (15 mm away from the tumor) of the ADC sample from FIG. 2A.

FIG. 2C shows the magnified representative image of the cytoplasm staining of adenocarcinoma tumor cells from FIG. 2A.

FIG. 3A shows a representative staining image of the dysplasia cells of a CIN3 tissue using a mouse monoclonal anti-HPV E7 antibody in an IHC assay according to another embodiment of the invention.

FIG. 3B shows the magnified representative image of the dysplasia epithelium of FIG. 2A, indicating specific nuclear staining of the CIN3 dysplasia.

FIG. 4A shows a representative staining image of the dysplasia cells of CIN2 tissues using an anti-HPV E6 mouse monoclonal antibody in an IHC assay according to one embodiment of the invention.

FIG. 4B shows a representative staining image of the normal epithelium adjacent to the dysplasia tissue of the CIN2 sample of FIG. 1A using the same anti-HPV E6 mouse monoclonal antibody an IHC assay.

FIG. 4C shows the staining results of dysplasia epithelium of a CIN3 tissue using the same anti-HPV E6 mouse monoclonal antibody as the one in FIG. 1A in an IHC assay according to another embodiment of the invention.

FIG. 4D shows the staining results of dysplasia epithelium of another CIN3 tissue using the same anti-HPV E6 mouse monoclonal antibody as the one in FIG. 1C in an IHC assay according to another embodiment of the invention.

FIG. 5: Sandwich assay/EIA Antigen test to detect the presence of the E6, E7 oncoproteins, and L1 viral proteins in the serum sample

DETAILED DESCRIPTION

Developing appropriate assays, such as HPV immunological assays, is needed for detection of HPV oncoproteins and identification of biomarkers for cervical cancer. Embodiments of the invention provide immunoassays, and kits for performing HPV immunoassays to detect HPV proteins in a biological sample. The invention also provides the use of the HPV immunoassays to identify suitable biomarkers for HPV related cancers, including cervical cancer. It is contemplated that, as evidenced by performing the immunological assays using the antibodies as described herein, the presence of HPV proteins, such as E6/E7 oncoproteins in CIN 2 and CIN 3 lesions could be used as indicator/biomarker to indicate high risk of cancer progression. Because of limited antibody available for the detection of these E6/E7 oncoprotein in situ, the invention also provides immunoassays, such as immunohistochemistry (IHC) assay, ELISA (enzyme linked immunoabsorbant assays), and kits for performing immunohistochemistry assay to detect the presence of HPV proteins in situ. In addition, cervical cancer biomarkers are identified, and methods and kits for identifying suitable invasive cervical cancer biomarkers and/or predicting the risk for malignant transformation into cervical cancer.

In one embodiment, an immunological assay is conducted on a clinical sample of a human subject to stain a nuclear portion of a human cell from the clinical sample using one or more monoclonal antibodies capable of binding to HPV early viral proteins. In another embodiment, staining of the nuclear portion of the human cells from the clinical sample is compared with the staining of the cytoplasmic portion of the human cells. Accordingly, performing the immunological assay of the invention provides evidence that positive staining of the nuclear portion correlates with the progression to a disease stage, including an early dysplasia stage, low-grade squamous intracervical lesion (LSIL), high-grade squamous intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2, CIN3) and combinations thereof. In addition, a correlation is found between positive staining of a cytoplasmic portion of the human cell and papillomavirus infection progressed into a disease stage, including a late dysplasia stage, cervical intraepithelial neoplasm (CIN3), invasive cervical cancer, squamous cell carcinoma (SCC), adenocarcinoma, and combinations thereof.

For example, a method of papillomavirus infection detecting a human subject includes conducting an immunohistochemistry assay on the slide containing a thin section of human tissue to detect in situ one or more papillomavirus proteins from one or more papillomavirus types present in the biological sample on the slide and using one or more anti-HPV antibodies to stain the thin section of human tissue. In one embodiment, the one or more anti-HPV antibodies may be anti-HPV monoclonal antibodies, for example, anti-HPV E6 monoclonal antibodies, anti-HPV E7 monoclonal antibodies, anti-HPV L1 monoclonal antibodies, and combinations thereof

In one aspect, the one or more monoclonal antibodies are generated against one or more purified recombinant papillomavirus proteins. In another aspect, the one or more anti-HPV antibodies are capable of recognizing a papillomavirus early viral proteins. In still another aspect, the anti-HPV antibody are capable of binding to HPV late viral proteins. The papillomavirus early protein may be, for example, HPV-16 E6 protein, HPV-16 E7 protein, HPV-18 E6 protein, HPV-18 E7 protein, and combinations thereof. The one or more purified recombinant papillomavirus proteins may include, but are not limited to, recombinant papillomavirus E6 protein, recombinant papillomavirus E7 protein, recombinant papillomavirus L1 protein, such as recombinant HPV-16 E6 proteins, recombinant HPV-16 E7 proteins, recombinant HPV-18 E6 proteins, recombinant HPV-18 E7 proteins, and HPV-16 L1 proteins, recombinant HPV-18 L1 proteins, and combinations thereof.

As another example, an immunohistochemistry assay can be performed on a clinical sample of the human subject using two or more antibodies, where each antibody is capable of binding to a HPV E6, E7, or L1 viral protein. As the expression levels of most HPV viral proteins, including E6, E7, and L1 viral protein in infected human subjects are extremely low, most clinical samples may escape detection by a single anti-HPV antibody. Thus, two or more anti-HPV antibodies can be used, and the staining of the human cells from clinical human subjects by the two or more antibodies are compared such that positive staining of the human cells by at least one of the two or more antibodies indicates papillomavirus infection in the human subjects. As an example, the anti-HPV antibodies pair may be a pair of an anti-HPV E6 antibody and an anti-HPV E7 antibody. As another example, the pair of two or more antibodies may be an antibody capable of binding to an early HPV viral protein and an antibody capable of binding to a late HPV viral protein.

Using the two or more anti-HPV antibodies, staining of the nuclear portion of the human cells from the clinical samples can be compared with the staining of the cytoplasmic portion, such that positive staining of the cytoplasmic portion of the epithelial tissue sample indicates dysplasia progression by HPV infection. Thus, two or more anti-HPV antibodies can be used in an immunohistochemistry assay to detect HPV infection at various disease stages, such as early stage HPV infection, late stage HPV infection, early stage cervical cell lesion, late stage cervical cell lesion, low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), cervical intraepithelial neoplasm stage 1, (CIN1), cervical intraepithelial neoplasm stage 2 (CIN2), cervical intraepithelial neoplasm stage 3 (CIN3), developed cervical cancer, adenocarcinoma (ADC), and squamous cell carcinoma (SCC).

In addition, a cytological papanicolaou smear assay can also be performed on the clinical sample to compare the results of the cytological papanicolaou smear test with the results of the one or more immunohistological assays. The resets of the papanicolaou smear assay are usually score and designated as follows:

(1) NILM: negative for intraepithelial lesion or malignancy; used when there is no cellular evidence of neoplasia; including microorganisms and/or other non-neoplastic findings such as reactive/ reparative changes;

(2) ASC-US: atypical squamous cells of undetermined significance; cells are usually the size of intermediate or superficial squamous cells and have nuclear changes that are suggestive but not diagnostic of LSIL or SIL not otherwise specified;

(3) ASC-H: atypical squamous cells cannot exclude HSIL; cells are usually the size of metaplastic cells and may be seen singly or in clusters; suggestive but not diagnostic of HSIL;

(4) LSIL: low grade squamous intraepithelial lesion, encompassing HPV cytopathic effect/ mild dysplasia/CIN 1;

(5) HSIL: high grade squamous intraepithelial lesion, encompassing: moderate dysplasia/CIN 2 and severe dysplasia/CIS/CIN 3 and HSIL with features suspicious for invasion. High grade squamous intraepithelial lesion or HSIL or HGSIL indicates moderate (CIN2) or severe (CIN3) cervical intraepithelial neoplasia or carcinoma in situ. It is usually diagnosed following a pap test. In some cases these lesions can lead to invasive cervical caner, if not followed appropriately. HGSIL does not mean that cancer is present. Of all women with HGSIL results, 2% or less have invasive cervical cancer at that time, however about 20% would progress to having invasive cervical cancer without treatment. To combat this progression, HGSIL is usually followed by an immediate colposcopy with biopsy to sample or remove the dysplastic tissue. This tissue is sent for pathology testing to assign a histologic classification that is more definitive than a Pap smear result. HGSIL generally corresponds to the histological classification of CIN2 or CIN3. Therefore, it is helpful to provide HPV IHC assay along with HE stain (hematoxylin and eosin stain) or HPV Immunocytological (ICC) assay along with the pap smear test for detecting HPV proteins in situ, particularly helpful in CIN2/CIN3 samples;

(6) Squamous cell carcinoma (SCC); cancer of the cervix, locally invasive into neighboring tissues, blood vessels, lymph channels and lymph nodes. In its advanced stages it can be difficult to treat and may prove fatal. Depending on the stage or degree of invasion, invasive cancer of the cervix may be treated with local excision, hysterectomy, radical hysterectomy, radiation, and chemotherapy;

(7) Adenocarcinoma: While most cancer of the cervix comes from the squamous cells making up the exterior skin, there is an occasional cancer that arises from the mucous-producing cells which line the endocervical canal leading up into the uterus. This glandular-type is called “adenocarcinoma” as opposed to “squamous cell carcinoma.” Adenocarcinoma can be difficult to detect. Unlike squamous cell cancer: Adenocarcinoma precursers, when present, can be difficult to identify on Pap smears. The slow progression of squamous cell dysplasia into squamous cell cancer of the cervix is not as uniform in adenocarcinoma.

Further, nucleic acid hybridization assay can also be performed on the clinical sample from a human subject to detect the presence of a papillomavirus genome in the clinical sample. Further, a hematoxylin and eosin staining assay may also be performed on the same set of clinical samples and the results of the immunohistochemistry assays or any additional immunological assays as described herein are compared with the results of the hematoxylin and eosin stainin assay.

One embodiment of the invention provides various novel monoclonal antibodies against HPV proteins, useful as biomarkers and useful tools for detecting HPV viral proteins, HPV oncoproteins, early screening of cervical cancer, and diagnosing CIN and/or invasive cervical and other cancers, are provided. The tools as provided herein can also be used in early clinical screening for HPV infection and general diagnosis for cervical cancer and other cancers, specific detection of invasive cervical cancer, detection of other HPV related cancers, early stage precancerous lesions as well as late stage cancer progression.

A kit for performing HPV IHC assay is also provided. The kit may include an pre-antibody blocking solution, post-antibody blocking solution, an anti-HPV antibody as the primary antibody, an anti-mouse or anti-rabbit immunoglobulins conjugated with HRP or biotin, or other agents as secondary antibody, a solution containing appropriate agents used as substrate for the secondary antibody to be detected. The anti-HPV antibody may be, for example, an anti-HPV E7 monoclonal antibody, an anti-HPV E6 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E7 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E6 monoclonal antibody, a combination of an anti-HPV E6 antibody and anti-HPV E7 monoclonal antibody. Such a kit can be used to perform an immunological assay, including, but not limited to, ELISA (enzyme linked immunoabsorbant assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, protein chip assays, radioimmunoprecipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays, immunohistochemistry for tissues and/or cervical cells, and immunocytological assays followed by flow cytometry, and combinations thereof.

One of embodiment provides various monoclonal antibodies against HPV viral proteins such that infection by high risk and low risk HPV types can be detected by a single monoclonal antibody. The invention also provides HPV type specific monoclonal antibodies for detecting only the high risk HPV types. The one or more papillomavirus types include high risk HPV types, low risk HPV types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, and combinations thereof.

In another embodiment, the immunological assay is used to detect a diseased stage caused by HPV infection. The disease stage may be, for example, an early stage HPV infection; a late stage HPV infection; low grade of squamous intraepithelial lesion (LSIL); high grade of squamous intraepithelial lesion (HSIL); Atypical squamous cells of undetermined significance (ASCUS); Atypical squamous cells without excluding HSIL (ASC-H); Atypical glandular cells (AGC); ervical intraneoplasm CIN1, CIN2, CIN3 representing a mild, moderate, or severe cell dysplasia. Respectively; invasive cervical cancer; adenocarcinoma (ADC); or squamous cell carcinoma (SCC).

The binding of the antibody with the one or more proteins from one or more papillomavirus types present in the biological sample were examined under a microscope, detecting the presence of an agent reacting with the tagged one or more antibodies, wherein the agent consists of a colormetric agent, a fluorescent chromogen, and combinations thereof. The biological sample consists of cervical cells, cervical tissues, cervical swabs, body fluids, serum, blood, tumors, cell cultures, biopsies, and combination thereof. The biological sample can be obtained from a group of people as general population for routine screening of cervical cancer.

Methods of producing the monoclonal antibody are provided herein to obtain monoclonal antibodies recognizing one or more common epitopes of HPV proteins among various HPV types. In addition, some of the monoclonal antibodies obtained herein are HPV type-specific while some of the monoclonal antibodies obtained herein are non-HPV type-specific. The non-HPV type-specific antibodies recognize most of the prevalent HPV types present in clinical samples; as a result, these monoclonal antibodies are suitable to be used in an assay screening for HPV infection in one or more clinical samples. Epitope mapping of these non-HPV type specific antibodies identifies and allocates the common epitope of the HPV specific proteins for binding of these monoclonal antibodies with most of prevalent HPV types in the clinical samples. These monoclonal antibodies are suitable for use as potential drug candidates for treatment of most HPV infections or cervical cancer. Furthermore, the common epitope(s) of these particular antibodies also present(s) the potential binding site to target E6 or E7 oncoproteins, or L1 viral proteins in drug design, drug screening and/or drug development.

For example, a number of cervical biopsy samples are tested in an immunohistohistochemistry (IHC) assay concurrently as a tissue microarray format using a monoclonal antibody to detect HPV proteins from a variety of HPV types (as confirmed by HPV DNA genotyping). Using a monoclonal antibody against HPV oncoprotein E7, the invention provides detection of the presence of HPV E7 protein in clinical samples having either single HPV infection or multiple HPV infections. A single anti-E7 monoclonal antibody as described herein can detect single HPV infection by at least HPV-6, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, etc., which are cancer-related HPV types (either high risk HPV types or low risk HPV types). A single anti-E7 monoclonal antibody can detect HPV infection by two or more HPV types, such as the combination of HPV-16, HPV-18, HPV-52, HPV-58, HPV-44, HPV-51, HPV-39, HPV-59, etc., which include high risk, low risk, and non-oncogenic α-papillomaviruses.

In addition, the monoclonal antibodies generated using methods of the invention are useful to detect infection by oncogenic HPVs, such as infection by high risk HPV types and/or low risk HPV types. As an example, antibodies raised against a recombinant protein HPV16 E6 oncoprotein generated by the method of invention are able to recognize E6 proteins present inside the cells of clinical samples due to single or multiple HPV infection, and react with E6 proteins from high risk HPV types (such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, etc.) or low risk HPV types (HPV-6, etc.). In addition, a single anti-E6 monoclonal antibody can detect multiple HPV infection in a clinical sample, having two or more HPV types, such as the combination of HPV-16, HPV-18, HPV-51, HPV-52, HPV-58, among others.

As another example, antibodies raised against a recombinant protein HPV16 L1 capsid protein generated by the method of invention are able to recognize L1 proteins present inside the cells of clinical samples due to HPV infection, and react with L1 proteins from high risk HPV types (such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, etc.) or low risk HPV types (HPV-6, etc.). In addition, a single anti-L1 monoclonal antibody can detect multiple HPV infection in a clinical sample, having two or more HPV types, such as the combination of HPV-16, HPV-18, HPV-51, HPV-52, HPV-58, among others.

In another embodiment, various monoclonal antibodies against HPV proteins, E6, E7 or L1 are provided, including those monoclonal antibodies specific for detecting HPV types correlated with the immunogens that the antibodies were raised, and other non-HPV type-specific monoclonal antibodies. In still another embodiment, monoclonal antibodies are provided in a screening/diagnosis assay with high positive predictive value (PPV) and high negative predictive value (NPV) for HPV associated CIN and invasive cervical cancer diagnosis. The high positive predictive value (PPV) in an assay represents statistically the possibility of relating CIN or invasive cancer to the detection of HPV associated proteins E6, E7, or L1 in the assay is very high, and vice versa. The high negative predictive value (NPV) in an assay represents statistically the possibility of relating CIN or invasive cancer to the non-detection of HPV associated proteins E6, E7, or L1 in the assay is very high, and vice versa. The antibodies of the invention includes, but are not limited to anti-E6, anti-E7, and anti-L1 antibodies, etc., and are used in one or more immunological assays to result in high PPV and/or high NPV values on clinical samples. For examples, samples confirmed with various grades of epithelial lesions (CIN2, CIN3, LSIL, HSIL, ASCUS) as well as different cervical cancers, squamous cell carcinoma (SCC, a type of common cervical cancer) and adenocarcinoma (ADC, a type of gland cancer) can be tested.

The PPV (positive predictive values) of the anti-E6 antibodies in an immunoassay as performed herein range from about 57% to about 100% for CIN2 samples, about 68% to about 100% for CIN3 samples, about 62% to about 100% for SCC samples, and about 63% to about 100% for ADC samples. The NPV vlaues of the antibodies of the invention range from about 54% to about 61% for CIN2 samples, about 60% to about 68% for CIN3 samples, about 60% to about 100% for SCC samples, and about 75% to about 100% for ADC samples. In addition, the specificity of the antibodies of the invention ranges from about 36% to about 100% for CIN2 samples, about 77% to about 100% for CIN3 samples, about 55% to about 100% for SCC samples, and about 50% to about 100% for ADC samples. The sentitivity of the antibodies of the invention ranges from about 17% to about 72% for CIN2 samples, about 30% to about 57% for CIN3 samples, about 67% to about 100% for SCC samples, and about 75% to about 100% for ADC samples

For example, one or more anti-E7 monoclonal antibody as provided herein are useful for detecting HPV infection and predicting HPV associated CIN or invasive cervical cancer in an immunoassay with a positive predictive value (PPV) ranging from about 57% to about 100%. The obtained monoclonal antibodies can be very useful in screening clinical samples for invasive cervical cancer. In an IHC assay on cervical biosy samples, the best results using the monoclonal antibodies of the invention result in about 100% positive predictive value (PPV) for invasive cervical cancer or at least more than 90%, considering the non-result from difficulties due to experimental procedural error. Also, monoclonal antibodies with about 100% positive predictive value (PPV) for IHC staining of CIN 2 or CIN3 clinical samples can also be obtained.

In another embodiment, a negative predictive value (NPV) ranging from more than about 50% to about 100% for clinical samples can be observed in an IHC assay for one or more anti-E7 monoclonal antibody. NPV value of about 100% for SCC and ADC clinical biopy samples as observed for some of the antibodies provided herein supports the use of these antibodies for diagnozing and screening HPV associated CIN or invasive cervical cancer.

On the cellular level, the HPV proteins can be observed in the nucleus and cytoplasm, but not in the membrane of a cell. It is found that the HPV proteins are present in the nucleus and/or cytoplasm of dysplasia epithelium from most of the samples tested. The staining of dysplasia cells by the antibodies of the invention results in diffused staining for the full layer of epithelium (the whole layer in thickness). However, there is focused nucleus staining in the basal, parabasal, and intermediate layer of the adjacent normal epithelium. It indicates that HPV proteins present in the peripheral of epithelium where HPV infection occurs adjacent to the dysplasia cells. However, HPV proteins are localized differently in adjacent normal epithelium and dysplasia epithelium.

For invasive cancer like SCC and ADC described here in this invention, staining of the dysplasia epithelium by anti-HPV protein antibodies displays distinct high levels of staining on both the nucleus and cytoplasm as compared to the staining of the adjacent normal epithelium. For HSIL like CIN 3 cases, about 90% to 100% of CIN 3 cases display distinct high levels of staining on the nucleus, while about 60% of those display distainct cytoplasmic staining. These data confirm that E6 or E7 expression is detected in most of HSIL like CIN3 and invasive cancer. We have also found that staining of the cytoplasm is only present in the dysplasia cells, but not in the adjacent normal epithilia.

For CIN2 cases, about 60% to 70 or 80% of CIN 2 cases display distinct high levels of staining by anti-HPV proteins antibodies on the nucleus, while about only 40% to 50% of those diaplay distinct cytoplasmic staining. Since CIN2 cases represent moderate HSIL, it's reasonable to explain CIN2 has lower rate of dysplasia progression, thus the cytoplasm staining by anti-E6 or E7 antibodies has less positive rate compared to CIN3 or invasive cancer. This method thus provides tool to distinguish dysplasia progression in HSIL. Therefore, staining of cytoplasm by anti-HPV oncoproteins antibody identifies progression of HSIL dysplasia cells.

Since 100% of invasive samples are positive in both cytoplasmic and nucleus staining by anti-HPV proteins antibodies, staining of both nucleus and cytoplasmic of dysplasia cells from CIN2/3 could indicate possibility of further progression. It is contemplated that the cytoplasmic staining is a unique staining pattern in localization of HPV proteins for those with potential to progress further to severe dysplasia or invasive cervical cancer. Thus, the cytoplasmic staining of dysplasia cells is a good tool to screen HPV associated cancer pathogenesis and cancer progression.

Comparing various HPV proteins related to HPV early genes and late genes, it is found that HPV E7 oncoprotein is present at high level than other HPV proteins such that it may be relatively easier for antibodies of the invention to detect HPV E7. In addition, HPV E7 can be served as a cervical cancer biomarker and the invention provides various antibodies tested to high PPV and NPV values, high specificity and sensitivity for clinical samples form all stages of CIN or cervical cancer development such that it is possible to use one single monoclonal antibody to detect HPV E7 proteins present in various early precancerous lesions as well as late stage invasive cancer progression. For example, the invention provides antibodies useful for in situ detection screening of clinical samples with early stage epithelium lesions, including CIN1 ASCUS, LSIL, HSIL, among others. In addition, the antibodies are also very successful in screening CIN2 and CIN 3 lesions as well as various types of cervical cancers during cancer development.

Detection of HPV DNAs, genomes, early viral proteins, late viral proteins, oncoproteins, and/or capsid proteins from various HPV genotypes can be performed by the method and detection assays as described herein and can be very useful in general clinical screening for HPV infection. Detection of HPV antibodies and/or oncoproteins by immunological assays can be used in early clinical screening for HPV infection and general diagnosis for cervical cancer and can be performed in a single rapid test or in multiplexed test. Comparative detection of altered levels of HPV proteins and host proteins can be performed in the same or different assays. It can also be used in diagnosing HPV-associated carcinomas of the uterine cervix, as well as those cases associated with epithelial cell abnormalities induced by HPV infection, pre-malignant and malignant HPV-associated epithelial cell lesions, and those at risk of developing HPV-associated cervical carcinoma and adenocarcinoma. The methods as described herein can be used independently or as an adjunct screening tool to convention cytological papanicolaou smear tests or histological tests and the results thereof can be compared for follow-up patient management.

In one embodiment, a method of screening a human subject of papillomavirus infection includes obtaining a clinical sample from the human subject, and conducting one or more immunological assays on the clinical sample from the human subject using various HPV recombinant proteins and lab-generated antibodies specific for HPV oncoproteins in order to detect and screen for the presence of HPV infection from the presence of HPV proteins and HPV antibodies in the human subject. In another embodiment, the HPV proteins in the human subject are detected using antibodies raised against HPV recombinant proteins, including but not limiting to various polyclonal and monoclonal antibodies against various HPV early and late proteins.

The one or more immunological assays as developed herein lend themselves to the high quality and properly purified recombinant proteins encoded by HPV early and late genes, as well as high quality polyclonal and monoclonal antibodies, resulting in immunological assays with very high sensitivity and specificity for screening HPV infection. The one or more immunological assays include, but are not limited to, protein chip assays, antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins ELISA (enzyme linked immunoabsorbant assays), assays for papillomavirus immunocomplexes, radioimmunoprecipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays, immunohistochemistry for tissues and/or cervical cells among others, and immunocytological assays followed by flow cytometry. The one or more immunological assays may be non-invasive with minimal or no additional instrument required.

The basic techniques for conducting the immunological assays can be found in “Antibodies: A Laboratory Manual”, Harlow and Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The basic techniques for conducting the immunological assays can be found in “Antibodies: A Laboratory Manual”, Harlow and Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989; “Molecular Cloning”, A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989, and others books and manuals known in the art. The related immunological assays, immunohistochemistry for tissues and/or cervical cells, and/or immunocytological assays followed by flow cytometry can also be found in co-pending U.S. patent applications: Ser. No. 11/559,366, filed on Nov. 13, 2006, titled “Detection method for human papillomavirus (HPV) and its application in cervical cancer”; U.S. Ser. No. 12/082,740, filed Apr. 14, 2008, titled “Protein chips for HPV detection”; Ser. No. 61/131,991, filed Jun. 13, 2008 titled “Antibodies and assays for HPV detection”; Ser. No. 61/192,912 Filed on Sep. 22, 2008, titled “Novel monoclonal antibodies against HPV proteins useful for early stage and late stage detection, screening, and diagnosis of HPV related cervical cancer”; Ser. No. ______ (NEOD/0004), filed concurrently as this application, titled “Novel monoclonal antibodies against HPV proteins”; Ser. No. ______ (NEOD/0005.01), filed concurrently as this application, titled “in situ detection of early stages and late stages HPV infection”; Ser. No. ______ (NEOD/0005.03), filed concurrently as this application, titled “Detection of early stages and alte stages HPV infection”. All of the above referenced applications are herein incorporated by reference.

EXAMPLES

1. Expression, Purification, and Preparation of HPV Recombinant Proteins to be used as Immunogens for Generating Antiserum and Anti-HPV Antibodies, and Screening Hybridoma Cell Lines for Monoclonal Antibodies

HPV recombinant proteins can be any kinds of HPV proteins, HPV proteins of early genes and/or late genes, including, but not limited to, E2, E6, E7, L1, L2 and can be from various HPV types. Full-length E6, E7, and/or L1 polypeptide sequence, which have been found very difficult to obtain and purify due to undesirable aggregation during protein purification, protein instability, low levels of expression, low immunogenic responses of purified proteins. For example, many early E6 oncoproteins contain many cysteine amino acids and thus the correct topography of the E6 oncoproteins requires formation of many disulfide bonds properly. In addition, it was known that certain immunological assays using small peptides of early E6 and E7 proteins results in extremely low assay specificity and sensitivity and thus unsuitable as tools for clinical in vitro diagnostics. Thus, the invention provides recombinant proteins, such as recombinant hybrid proteins containing a partial sequence or a full length sequence of HPV oncogenic proteins.

1). Cloning and production of various recombinant proteins encoded by HPV16 E6 and HPV18 E6 gene. An exemplary oncogenic E6 early gene from an exemplary HPV type, HPV-16, was clone. The HPV 16 E6 gene cloned herein is a 474 base pair (b.p.) DNA fragment containing the 157 amino acid coding region of the whole HPV-16 E6 gene and obtained by polymerase chain reaction (PCR) amplification. The DNA sequence of the isolated DNA fragment was confirmed by comparing with the sequence from Gene Bank database. Recombinant HPV-18 E6 protein was also obtained. All cloning procedures are carried out according to the protocols described in “Molecular Cloning”, A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. In addition, HPV18 E6 gene was also cloned and the DNA sequence was confirmed.

2). Cloning and production of various recombinant proteins encoded by HPV16 E7 and HPV18 E7 gene. An exemplary oncogenic E7 early gene from an exemplary HPV type, HPV-16, was cloned. A 294 base pair (b.p.) DNA fragment containing the 99 amino acid coding region of the entire HPV-16 E7 gene was obtained by polymerase chain reaction (PCR) amplification. The DNA sequence of the isolated DNA fragment was confirmed by comparing with the sequence from Gene Bank database. Recombinant HPV-18 E7 protein was also obtained. In addition, E7 DNA fragments from different HPV types can also be cloned from different clinical samples or sources.

3). Cloning and Production of various recombinant Proteins encoded by HPV16 L1 and HPV18 L1 gene. An exemplary late gene from an exemplary HPV type, HPV-16, was cloned. A 1596 base pair (b.p.) DNA fragment containing the 531 amino acid coding region of the HPV-16 L1 gene was obtained by polymerase chain reaction (PCR) amplification. The DNA sequence of the isolated DNA fragment was confirmed by comparing with the sequence from Gene Bank database. In addition, L1 DNA fragments from different HPV types can also be cloned from different clinical samples or sources.

A recombinant N-terminal fragment of HPV 16 L1 protein was also obtained from a His-tagged expression system. The molecular weight of the HPV-16 L1 N-terminal recombinant protein ais bout 34 KD. L1 C-terminal fragments can also be obtained. Recombinant HPV-18 L1 protein was also obtained and used as immunogens for generating antiserum, polyclonal and monoclonal antibodies.

The one or more recombinant proteins as described herein can be expressed in various suitable systems, such as bacterial expression systems, viral expression systems, yeast expression systems, mammalian expression systems, e.g., in E coli, yeast, baculovirus, and/or mammalian cell cultures, generally known in the field. Although the polypeptides could be obtained by other means, embodiments of the invention provide one or more recombinant proteins mostly in (or close to) their native forms, which may be a much desirable conformation for binding with antibodies from tissues of human subjects with HPV infection in an immunological assay. For example, GST, MBP, or His tagged-HPV16-E6, HPV18 E6, HPV16 E7, HPV18 E7, HPV16 L1, and HPV18 L1 recombinant proteins were expressed in E. coli BL21(DE3) using IPTG driven induction. After induction of protein expression, tagged-HPV recombinant proteins were obtained from soluble fraction after lysis of the cultured cells and purified to a final concentration of about 0.1 to 1 mg/ml or higher. The purity of the recombinant HPV proteins was estimated to be>90% based on PAGE analysis. Recombinant HPV proteins were used to detect the presence of HPV antibody on clinical samples and was also used as immunogens for producing polyclonal antiserum and monoclonal antibodies.

The cell culture containing various recombinant papillomavirus proteins in various expression vectors as described herein are then scaled up to 1 liter or 10 liter, or 100 liters or higher to obtain high quantity of soluable recombinant protein for purification. The soluble fraction after cell lysis was passed through various chromatography columns with appropriate expression systems to bind to the tag expressed along with the HPV recombinant proteins. The tag-HPV recombinant proteins were then eluted from the column and concentrated down to 100 ml or 10 ml to 1 ml. The purified soluble recombinant HPV proteins were further concentrated and dailysed with buffers at neutral pH or PBS buffers to be used as immunogen to generate antiserum against the HPV proteins. The soluble recombinant HPV proteins were thus purified from soluble fractions and folded close to their native folding states as in vivo natural conditions.

Obtaining high quality purified recombinant HPV proteins is critical in generating various types of monoclonal antibodies that recognizing common epitopes or specific epitopes for detecting HPV infection. The purified recombinant HPV proteins were tested to confirm its binding to the HPV antibody from the HPV infected clinical samples. Thus, such purified recombinant HPV proteins are suitable for use as immunogen to raise antiserum and generate antibodies that can recognize natural HPV viral proteins in vivo.

2. Anti-HPV Polyclonal Antibody Production:

Recombinant HPV E6, E7 or L1 proteins expressed in E coli was purified, concentrated, and dialyzed with PBS to be used as immunogens. Immunization was followed by standard protocol. Titer of each serum obtained was tested by ELISA assays followed by periodical boosting and bleeding. Production bleed from optimal titer was collected; processed serum was used to do immunoglobulin (Ig) purification via protein A columns or affinity columns. Purified IgG was used as anti-HPV antibodies for HPV immunoassays.

Monoclonal antibodies, polyclonal antibodies, and antiserum were obtained, purified, and tested herein to be able to detect HPV infection regardless of the pathogenesis of HPV infection, cell lesions, inflammatory, or cancer disease development. Other researchers have tried to develop anti-HPV monoclonal antibodies but have failed because they failed to generate sufficient HPV proteins for monoclonal antibodies production; they failed to generate monoclonal antibodies with high specificity because the immunogens were not immunogenic; or the generated antibodies were not able to recognize native forms of HPV proteins present in clinical samples with early stage HPV infection. Some antibodies raised against mutant peptides were only able to recognize late stage cervical cancer, but are not sure whether their antibodies would recognize wild type HPV native proteins or any early stage HPV infection. In addition, late stage HPV detection is too late for disease intervention and treatment.

The clinical utility of the antibodies described herein was validated by HPV immunoassays, such as ELISA assays, immunocytochemistry assays, immunohistochemistry assays, using appropriate clinical samples. The novel monoclonal antibodies and antiserum, obtained from methods of this invention are able to interact and bind HPV viral proteins present in clinical samples, which have been confirmed to contain early stage cell lesions such as cervical intraepithelial neoplasia (CIN) as well as late stage HPV associated cervical cancer. The monoclonal antibodies and antiserum as described herein provide powerful tools to detect and screen HPV related pathogenesis and cervical cancer development in both early stages and late stages; thus provides an avenue to intervene disease progression and a chance to provide early treatment.

3. HPV Monoclonal Antibody Development:

Recombinant HPV E6, E7 or L1 proteins expressed in E coli was purified, concentrated, and dialyzed with PBS to be used as immunogen. Immunization of mice was followed by standard procedure. Titer of the obtained serum was tested by ELISA followed by periodical boosting and bleeding. When the titer of the serum of the mice reaches optimal, fusion of the spleen cells of the mice with tumor cells was done by standard procedure. Clones of fused cells, e.g., hybridoma cells, were further cultured.

1). Hybridoma screening: To obtain anti-HPV antibody producing hybridoma cells with pan and specific binding capability to various HPV proteins as described in this invention, hybridoma clones were screened with various proteins, including, not only the original immunogens but also additional HPV proteins as positive screening, and unrelated proteins as negative screenings. For example, two or more purified HPV recombinant proteins were used to screen against each hybridoma clone to screen and obtain monoclonal antibody-producing hybridoma cell lines and to test and understand the specificity of each antibody-producing hybridoma cell line thus obtained.

As an example of hybridoma screening, antibody-producing hybridoma cells were screened with two or more purified recombinant human papillomavirus proteins such that the monoclonal antibody is capable of reacting with the two or more purified recombinant human papillomavirus proteins. The two or more purified recombinant human papillomavirus proteins include, but are not limited to, HPV 16 E6 protein, HPV 16 E7 protein, HPV 16 L1 protein, HPV 18 E6 protein, HPV18 E7 protein, HPV 18 L1 protein, and other HPV early proteins and late proteins from various HPV types.

The antibody-producing hybridoma cells were screened with positive reactivity to all of the two or more purified recombinant human papillomavirus proteins and negative reactivity to non-HPV proteins, including BSA, his₆ tags, GST proteins, maltose binding proteins (MBP), other tags or proteins used in recombinant protein, and other readily available non-HPV proteins. As such, the monoclonal antibodies generated form such hybridoma screening is capable of binding to all of the two or more HPV viral proteins (e.g., the HPV viral proteins present in clinical samples), which correspond to the two or more purified recombinant human papillomavirus proteins.

One example of the two or more purified recombinant human papillomavirus proteins are HPV early proteins such that the monoclonal antibody is capable of reacting with the two or more human papillomavirus early proteins. For example, one hybridoma cell line thus screened and obtained can produce a monoclonal antibody recognizing a common epitope on both HPV16 E6 and HPV16 E7 proteins. Another hybridoma cell line thus screened and obtained can produce a monoclonal antibody recognizing a common epitope on both HPV18 E6 and HPV18 E7 proteins.

Another example of the two or more purified recombinant human papillomavirus proteins includes a purified recombinant human papillomavirus early protein and a purified recombinant human papillomavirus late protein such that the monoclonal antibody produced is capable of reacting with a common epitope on the purified recombinant human papillomavirus early protein and the purified recombinant human papillomavirus late protein. The purified recombinant human papillomavirus early protein may be HPV 16 E6 protein, HPV 16 E7 protein, HPV 18 E6 protein, HPV18 E7 protein, and other HPV recombinant early proteins, and the purified recombinant human papillomavirus late protein may be HPV 16 L1 protein, HPV 18 L1 protein, and other HPV recombinant late proteins. For examples, hybridoma cell lines thus screened and obtained can produce a monoclonal antibody recognizing a common epitope on HPV16 E6, HPV16 E7, and HPV16 L1 proteins; or a monoclonal antibody recognizing a common epitope on HPV16 E6 and HPV18 E6 proteins; or monoclonal antibody recognizing a common epitope on HPV16 E7 and HPV18 E7 proteins; or monoclonal antibody recognizing a common epitope on HPV16 E6, HPV16 E7, HPV16 L1, HPV18 E6, and HPV18 E7 proteins. More examples are provided in the drawings of this invention.

The antibody-producing hybridoma cells were also screened with a first purified recombinant human papillomavirus protein from a first HPV type and a second purified recombinant human papillomavirus protein from a second HPV type such that the monoclonal antibody is capable of reacting with a common epitope on human papillomavirus proteins from two or more different HPV types. The first and the second HPV types can be HPV 16, HPV 18, and other HPV types. The two or more different HPV types can be, for example, high risk HPV types, low risk HPV types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56. As an example, the first and the second purified recombinant human papillomavirus proteins may be recombinant HPV 16 E6 protein, recombinant HPV 16 E7 protein, recombinant HPV 16 L1 protein, recombinant HPV 18 E6 protein, recombinant HPV18 E7 protein, and recombinant HPV 18 L1 protein.

As another example of hybridoma screening, antibody-producing hybridoma cells were screened with positive reactivity to some of the two or more purified recombinant human papillomavirus proteins and negative reactivity to some of the two or more recombinant human papillomavirus proteins and/or non-HPV proteins. As such, the monoclonal antibodies generated form such hybridoma screening is capable of binding to some HPV viral proteins but not other HPV viral proteins.

For example, a monoclonal antibody is obtained by screening antibody-producing hybridoma cells with a first purified recombinant human papillomavirus protein from a first HPV type and a second purified recombinant human papillomavirus protein from a second HPV type such that the monoclonal antibody is capable of reacting with a specific epitope on only one of the first and the second purified recombinant human papillomavirus proteins and not the other purified recombinant human papillomavirus protein. Specific monoclonal antibodies obtained includes a monoclonal antibody capable of binding to only HPV 16 E6 protein, but not any other HPV proteins; a monoclonal antibody capable of binding to only HPV 16 E7 protein, but not any other HPV proteins; a monoclonal antibody capable of binding to only HPV 16 L1 protein, but not any other HPV proteins; a monoclonal antibody capable of binding to only HPV 18 E6 protein, but not any other HPV proteins; and a monoclonal antibody capable of binding to only HPV 18 E7 protein, but not any other HPV proteins.

2). Hybridoma cell line stocks: Hybridoma cell line clones with desired positive reactivity and desired negative reactivity as judged by an immunoassays (e.g., ELISA, EIA and other assays) were selected and cloned down to single cell. Each single cell clone was then grown up by tissue culture. When the cell numbers reach millions of cells per ml, the cells were frozen down and kept at −80° C. or in liquid nitrogen as storage stocks.

3). Ascites Production: Each hybridoma cell line was grown in tissue culture and injected to mice for ascites production. Ascites were collected and processed for immunoglobin purification by protein G columns. Purified immunoglobin from each hybridoma cell line was isotyped and used for HPV immunoassays.

4. HPV Immunohistochemistry (IHC) Assay: 1). HPV IHC Kit and the Assay:

In one embodiment, a kit for performing a HPV IHC assay is provided. The kit may include an antigen retrieval agent, a pre-antibody blocking solution, a post-antibody blocking solution, an anti-HPV antibody as the primary antibody, an anti-mouse or anti-rabbit immunoglobulins conjugated with HRP or biotin, or other agents as secondary antibody, a solution containing appropriate agents used as substrate for the secondary antibody to be detected.

The antigen retrieval agent may contain a solution in low pH, or neutral pH or high pH buffer. The pre-antibody blocking solution may contain certain proteins or BSA, or serum or other agents to block the cells from nonspecific binding of antibody. The post blocking solution may contain similar solution as the pre-antibody blocking solution with less proteins or serum to be used along with primary antibody incubation. The solution containing HPV antibodies may be in concentrated form, or may be in diluted form as ready to use reagent. The anti-HPV antibodies may also be directly tagged with HRP or biotin, or other agents to be detected following appropriate agents used as substrate. The solution containing secondary antibodies may be in concentrated form, or may be in diluted form as ready to use reagent. The solution containing appropriate agents used as substrate may include DAB (3.3′-diaminobenzidine) as one component, or two components, or AEC (3-Amino-9-Ethylcarbazole) substrate as one component, or two components, or other substrates.

Once the cervical tissues are processed and fixed, the Immunohistochemistry (IHC) assay is performed by boiling the tissues on the slide with antigen retrieval buffer for a period of time. The slides were then cool down to room temperature, blocked with pre-antibody blocking solution for a period of time, then incubated with the HPV antibodies. The slides were then washed 3 to 5 times with PBS or H2O, or other solution to remove any unbound HPV antibody. Then the slides were incubated with the secondary antibody, for example, anti-mouse IgG HRP, followed by appropriate substrate for detection. As an example, DAB is oxidized in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity. The precipitate may range in color from a light golden brown to dark golden brown depending upon the amount of enzyme present. The golden brown precipitate viewed under a microscope indicates the specific binding of HPV antibodies with HPV proteins present in the cells off the tissue section on the slide. The assay can be performed at room temperature or higher temperature to accelerate the binding reaction. This HPV IHC assay can be performed manually, or operated by IHC automation, thus provides a powerful tool to detect HPV infection and HPV oncoproteins in situ. Therefore, the HPV IHC staining assay is very useful as a confirmatory test. For the dysplasia cells identified, HPV IHC staining may provide additional information for status of HPV infection and/or expression of HPV oncoproteins. In addition, overexpression of HPV E6 and E7 oncoproteins in various stage of cervical dysplasia may indicate progression of CIN and/or cervical cancer development.

2). Sample selection and preparation: In order to analyze if the anti-HPV antibody provided in this invention is able to detect HPV proteins in situ from different stage of CIN or cancers, the cervical tissues to be tested on the IHC assay include HSIL consisting of CIN2 (stage 2 of Cervical Intraepithelial Neoplasia with lesions appearing moderate) and CIN3 (stage 3 of Cervical Intraepithelial Neoplasia with lesions appearing severe), and invasive cancer consisting of squamous cell carcinoma (SCC, the most common carcinoma in cervical cancer) and adenocarcinoma (ADC, the gland type of carcinoma). Paraffin tissues blocks sectioned into 4 microns were placed on slide and baked at 60 C overnight. Deparaffin/hydrate sections were unmasked followed by standard IHC staining procedures. Purified monoclonal antibody against HPV proteins were diluted to use as the primary antibody. Staining procedure is followed by secondary antibody solution, washing, followed by appropriate substrate reagent to each section. As soon as the sections develop, immerse slides in dH₂O, counterstain sections with hematoxylin, dehydrate and mount coverslips.

3). Tissue Microarray: In order to perform homogeneous assay for many samples in one reaction, tissue microarray was generated to spot many samples on one slide. To process total of 84 samples from CIN2, CIN3, or invasive cancers, three tissue microarrays were prepared: One contains 30 individual CIN 2 and their peripheral normal epithelia, One contains 30 individual CIN 3 and their peripheral normal epithelia, One contains 12 cervical squamous cell carcinomas and their normal epithelial counterparts, and 12 adenocarcinomas and their normal epithelial counterparts, vaginal or cervical mucosa of at least 15 mm away from the gross tumor border. One representative tissue spot for neoplasia and another one spot representing its normal counterpart were taken for each CIN case. For the case of invasive cancer, 2 spots of tumor tissue and one spot of the normal counterpart were taken. A 2 mm round tissue spot was retrieved from the corresponding paraffin-embedded tissue block after taking a tissue slide for HPV DNA typing.

4). HPV DNA test: HPV DNA typing of each case was identified by a modified MY11/GP6+ PCR-based reverse-blot assay using EasyChip® HPV blot or a HR-HPV chip, which contained 13 type-specific oligonucleotides on a nylon membrane. Total cellular DNA was used as source of nucleic acid for amplification followed by hybridization for detection.

5). IHC score and data interpretation: The staining of each dot on the tissue microarray slide was viewed by certified anatomy pathologist under a microscope. Areas of tumor cells or dysplasia cells were looked up to find the percentage of cells stained, with staining intensity of score 0-3. Adjacent normal epithelium or normal tissue 15 mm away from its corresponding dysplasia or tumors was also scored. All data were scored by certified pathologist. Stained percentage 10% was used as cut off to determine positive or negative of the assay. All data were shown as Tables 1-17.

To demonstrate detection of HPV proteins in invasive cervical cancer, tissues of squamous cell carcinoma (SCC), appearing to be the most common cervical cancer, were processed to be performed on HPV IHC assay. FIG. 1A-1D show IHC staining of squamous cell carcinoma tissue using a mouse monoclonal HPV E7 antibody. FIG. 1A shows the representative image of the SCC tissue from tissue microarray stained by IHC using an anti-HPV E7 mouse monoclonal antibody. FIG. 1B shows the representative image of the normal epithelium (15 mm away from the tumor tissue) of the SCC subject from FIG. 1A. FIG. 1C shows the representative image of another SCC sample from tissue microarray stained by IHC using the same anti-HPV E7 antibody. FIG. 1D shows the magnified representative image of the tumor cells stained by the same anti-HPV E7 antibody in cytoplasm from FIG. 1C. Results indicate expression of E7 oncoprotein can be detected in the tumor cells of SCC tissue. Solid Black arrows indicate the specific staining of E7 protein in tumor cells, while empty clear arrows indicate the normal cells with no staining. These results demonstrate that in situ presence of HPV E7 protein from SCC cervical tissues can be detected by the mouse monoclonal anti-HPV E7 antibody used in the IHC assay.

To demonstrate detection of HPV proteins in another type of invasive cervical cancer, FIGS. 2A-2C show IHC staining of cervical adenocarcinoma using the same mouse monoclonal HPV E7 antibody. Results indicate expression of E7 oncoprotein can be detected in the tumor cells of adenocarcinoma tissue. Solid Black arrows indicate the specific staining of E7 protein in tumor cells, while empty clear arrows indicate the normal cells with no stain. FIG. 2A shows the representative image of the tumor cells of adenocarcinoma (ADC) sample stained by IHC using the same anti-HPV E7 monoclonal antibody shown in FIG. 1. FIG. 2B shows the representative image of the corresponding normal epithelium (15 mm away from the tumor) of the ADC sample from FIG. 2A. FIG. 2C shows the magnified representative image of the cytoplasm staining of adenocarcinoma tumor cells from FIG. 2A. Highly magnified images indicate localization of the E7 proteins expressed in the cytoplasm of tumor cells, but not in the normal epithelium, or other cells including stroma cells. These data demonstrate the IHC staining by the E7 monoclonal antibody can detect tumor cells of adenocarcinoma showing similar staining pattern found in SCC.

TABLE 1 IHC staining results (stained %) and HPV DNA typing for 12 SCC biopsy samples and 12 ADC biopsy samples (C: Cytoplasmic; N: Nucleus; Dys: dysplasia or tumor cells). Anti-E7 Anti- Another Anti- Normal Another E6 anti-E6 L1 Dys epith. (% anti-E7 Dys Dys. Dys. (% stained) stained) Dys (%) (%) (%) (%) Sample # HPV type C N C N C C C C SCC-1 18 85 85 0 20 12.5 10 70 55 SCC-2 16, 52 90 85 0 25 15 15 10 55 SCC-3 16 60 65 0 40 5 0 10 20 SCC-4 16 92 50 0 40 5 0 10 85 SCC-5 16, 52, 58 92 55 0 50 20 5 15 88 SCC-6 18, 52, 58 90 60 25 18 10 70 SCC-7 16, 52 92 75 0 30 30 5 10 20 SCC-8 16, 58 10 10 0 5 0 0 10 50 SCC-9 no DNA 95 60 0 40 25 8 15 8 SCC-10 18 92 65 0 60 45 25 20 65 SCC-11 16, 58 0 80 5 0 0 SCC-12 33 95 90 0 0 30 1 20 55 ADE-1 16, 18 30 20 0 50 15 25 20 82 ADE-2 no DNA 62 40 0 30 35 70 35 78 ADE-3 16 20 30 0 20 35 55 60 ADE-4 16, 18 80 80 0 0 10 5 0 90 ADE-5 51, 52 95 80 0 50 10 70 15 92 ADE-6 11, 16, 52 0 40 5 0 0 15 ADE-7 18 50 40 0 60 25 20 20 75 ADE-8 18 85 60 0 40 15 50 15 82 ADE-9 45 82 55 0 30 30 2 20 40 ADE-10 18 15 10 0 40 15 15 5 70 ADE-11 18, 59 70 0 0 50 15 8 5 65 ADE-12 18 30

To analyze the HPV IHC results from each subject of invasive cancer, Table 1 shows data from 24 cases of invasive cancer samples with IHC score for staining of cytoplasm (C), and nucleus (N) using C, or N followed by the % of staining using the anti-HPV E7 antibody shown in FIG. 1A-1D & FIG 2A-2C. Additional anti-HPV antibodies including another anti-E7 antibody, Anti-HPV E6 antibody like MAb1 and MAb 7 and anti-HPV L1 antibody were also also tested on the same tissue microarray. To demonstrate the IHC staining by various anti-HPV antibodies, IHC score from cytoplasm staining of tumor cells using other anti-HPV antibodies was also shown in Table 1. Results of HPV DNA typing were also shown on the table for its corresponding case.

As shown in Table 1, both nucleus and cytoplasmic staining are found in all the subjects of tumor cells from SCC and ADE stained by the anti-E7 antibody. However, there is more staining (percentage stained) found in the cytoplasm of tumor cells comparing the staining of nuclear of tumor cells. The detection of HPV E7 protein in its adjacent normal epithelium cells was only found in nucleus, but not found in the cytoplasm of the epithelial cells. The staining of cytoplasm appears most distinguishable in tumor cells compared to its corresponding normal adjacent cells. These data demonstrate expression of HPV E7 proteins was detected in the cytoplasm and nuclear of tumor cells of SCC and ADE tissues. The localization of the E7 proteins expressed in the cytoplasm of tumor cells, but not in the normal epithelium or stroma cells appears tumor specific. HPV E7 proteins present in the nucleus of normal adjacent epithelium and tumor cells detected by the anti-HPV E7 antibody indicate HPV infection with oncoproteins expression. Similar staining pattern was also found when used other anti-HPV antibodies as shown in Table 1. Data indicate that the HPV IHC assay as described herein can detect HPV early gene such as E6, E7, and late gene such as L1 proteins present in the tumor cells of cervical cancer tissues.

Comparing the results of HPV IHC to the HPV DNA typing, the anti-E7 antibody reacts positively with all the HPV types present in the samples tested. For example, the anti-E7 monoclonal antibody as described herein can detect single HPV infection by at least HPV-16, HPV-18, HPV-33, HPV-45, etc., which are cancer-related HPV types (high risk HPV types). The single anti-E7 monoclonal antibody can also detect HPV infection by two or more HPV types, such as the combination of HPV 11, HPV-16, HPV-18, HPV-52, HPV-58, HPV-51, HPV-59, etc., which include high risk, low risk, and non-oncogenic α-papillomaviruses. However, infection by multiple HPV types contains at least one type is high-risk HPV type. These data indicate that the anti-E7 antibody described in this invention is non-type specific, thus provides a powerful tool to detect HPV E7 proteins from most common high-risk HPV types in the cervical cancer.

To demonstrate detection of HPV proteins in non-invasive with severe dysplasia cervical tissue (HSIL; high grade squamous intraneoplasm lesion, stage 3), FIG. 3 show IHC staining of CIN3 tissue using the same mouse monoclonal HPV E7 antibody. Results indicate expression of E7 oncoprotein can be detected in the stage 3 of CIN tissue. Solid Black arrows indicate the specific staining of E7 protein in dysplasia cells, while empty clear arrows indicate the normal cells with no stain. FIG. 3A shows the representative image of the dysplasia cells of a cervical intraepithelial neoplasm (CIN3) tissue stained by IHC using the same anti-HPV E7 antibody shown in FIG. 1 and FIG. 2 for invasive cancer tissue. FIG. 3B shows the representative image of the adjacent normal epithelium of the CIN3 tissue of FIG. 3A. These results demonstrate that in situ presence of HPV E7 protein from CIN3 cervical tissues can be detected by the mouse monoclonal anti-HPV E7 antibody used in the IHC assay described herein.

To analyze the HPV IHC results from each subject of CIN3, Tabel 2 shows data from 30 cases of CIN 3 samples with IHC score for staining of cell membrane (M), cytoplasm (C), and nucleus (N) using M, C, or N followed by the % of staining with the anti-E7 antibody. Additional anti-HPV antibodies including Anti-HPV E6 antibody like MAb1 and MAb 7 and anti-HPV L1 antibody were also also tested on the same tissue microarray. To demonstrate the IHC staining by various anti-HPV antibodies, IHC score from cytoplasm staining of tumor cells using other anti-HPV antibodies was also shown in Table 1. Results of HPV DNA typing were also shown on the table for its corresponding case.

As shown in Table 2, nucleus staining are found in the dysplasia cells of all the CIN3 samples tested while only certain proportion of cases found staining of cytoplasm by the anti-E7 antibody. The results indicate that there is more staining found in the cytoplasm than in the nuclear of dysplasia cells. As shown previously in invasive cancer tissues, HPV E7 protein in its adjacent normal epithelium cells was only found in nucleus, but not found in the cytoplasm of the epithelial cells. The staining of cytoplasm appears most distinguishable in dysplasia cells compared to its corresponding normal adjacent cells. The localization of the E7 proteins expressed in the cytoplasm of dysplasia cells, but not in the normal epithelium or stroma cells appears HSIL specific. These data demonstrate expression of HPV E7 proteins can be detected in the cytoplasm and nuclear of dysplasia cells of CIN3 tissues. HPV E7 proteins present in the nucleus of normal adjacent epithelium and dysplasia cells detected by the anti-HPV E7 antibody indicate HPV infection with oncoproteins expression. For the cases with high level expression of HPV E7 proteins detected in the cytoplasm of dysplasia cells, it may suggest specific indication of dysplasia progression. Similar staining pattern was also found when used other anti-HPV antibodies as shown in Table 2. Data indicate that the HPV IHC assay as described herein can detect HPV early gene such as E6, E7, and late gene such as L1 proteins present in the dysplasia cells of CIN3.

Comparing the results of HPV IHC to the HPV DNA typing, the anti-E7 antibody reacts positively with all the HPV types present in the samples tested. For example, the anti-E7 monoclonal antibody as described herein can detect single HPV infection by at least HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-58, etc., which are cancer-related HPV types (high risk HPV types). The single anti-E7 monoclonal antibody can also detect HPV infection by two or more HPV types, such as the combination of HPV-16, HPV-18, HPV-33, HPV-39, HPV-52, HPV-58, etc., which include most common high-risk HPV. These data indicate that the anti-E7 antibody described in this invention is non-type specific, thus provides a powerful tool to detect HPV E7 proteins from most common high-risk HPV types in the CIN3 tissues.

TABLE 2 IHC staining results (stained % and score; 0-3) and HPV DNA typing of 30 CIN 3 samples (M: Membrane; C: Cytoplasmic; N: Nucleus; Dys: Dysplasia). anti-E7 Dysplasia Normal Anti-E6 Another Anti-L1 (% epithelium Dys. anti-E7 Dys. ID HPV stained) (% stained) (%) Dys. (%) (%) # type M C N M C N Cyto Cyto Cyto 31 33 0 80 80 0 0 50 70 40 80 32 16 0 80 80 60 0 0 5 33 16, 58 0 0 60 34 31 0 50 70 0 0 50 0 0 10 35 16, 39 0 70 90 0 0 40 0 10 30 36 31 0 70 60 0 0 50 0 20 20 37 39 0 0 40 0 0 0 0 0 0 38 16 0 0 40 39 16 0 60 70 0 0 40 0 0 40 58 0 90 90 0 0 50 50 0 30 41 16 0 0 50 0 0 50 0 20 20 42 16 0 70 70 0 0 30 0 0 43 33 0 0 90 0 0 50 0 0 5 44 52 0 70 80 0 0 50 70 10 50 45 51, 52 0 90 90 0 0 30 80 50 10 46 16 0 0 80 0 0 50 0 0 5 47 16 0 60 80 0 0 50 30 10 20 48 16, 58 0 0 80 0 0 50 0 0 10 49 31 0 80 60 50 70 40 40 50 16 0 0 60 0 0 30 0 20 20 51 6 0 0 20 0 52 16, 18, 0 0 20 0 0 30 0 0 0 33, 39 53 51, 52, 0 70 60 0 0 60 60 40 58 54 16, 45 0 0 70 0 0 50 0 20 20 55 16 0 0 75 0 0 50 0 0 0 56 33, 52 0 0 80 0 0 50 0 0 10 57 16 0 0 50 0 0 40 0 0 0 58 33 0 0 80 0 0 0 20 10 59 16 0 0 60 0 0 20 0 10 5 60 16, 52, 0 70 80 0 0 50 70 0 20 58

To demonstrate detection of HPV proteins in HSIL with moderate dysplasia (stage 2 of CIN), FIG. 4 show IHC staining of CIN2 tissue using the mouse monoclonal anti-HPV E6 antibody. Results indicate expression of E6 oncoprotein can be detected early in the stage of CIN2. FIG. 4A shows the representative image of the dysplasia cells of cervical intraneoplasm (CIN2) tissues stained by immunohistocytostaining (IHC) using the anti-E6 monolonal antibody. FIG. 4B shows the representative image of the adjacent normal epithelium from the dysplasia tissue of the CIN2 sample of FIG. 4A. FIG. 4C shows the representative image of the dysplasia epithelium of another CIN2 sample stained by IHC using the same anti-E6 monolonal antibody. FIG. 4D shows the magnified representative image of the dysplasia epithelium in FIG. 4C. Solid Black arrows indicate the specific staining of E6 protein in dysplasia cells, while empty clear arrows indicate the normal cells with no stain. Similar staining pattern of CIN2 found in CIN3 indicate localization of the E6 proteins expressed in the cytoplasm of dysplasia cells, but not in the normal epithelium, or other cells including stroma cells. These results demonstrate that in situ presence of HPV E6 protein from CIN2 cervical tissues can be detected by the mouse monoclonal anti-HPV E6 antibody used in the IHC assay described herein.

To analyze the HPV IHC results from each subject of CIN2, Table 3 shows data from 30 cases of CIN 2 samples with IHC score for staining of cell membrane (M), cytoplasm (C), and nucleus (N) using M, C, or N followed by the % of staining with the anti-E7 antibody. Additional anti-HPV antibodies including Anti-HPV E6 antibody like MAb1 and MAb 7 and anti-HPV L1 antibody were also also tested on the same tissue microarray. To demonstrate the IHC staining by various anti-HPV antibodies, IHC score from cytoplasm staining of dysplasia cells using other anti-HPV antibodies was also shown in Table 3. Results of HPV DNA typing were also shown in the table for its corresponding case.

TABLE 3 IHC staining results (stained % and score; 0-3) and HPV DNA typing for 30 biopsy samples (CIN2). (M: membrane; C: cytoplasmic; N: nucleus; Dys: dysplasia) Anti-E7 another Dysplasia Normal Anti-E6 anti-E7 Anti-L1 (% epithelium Dys. Dys. Dys. ID HPV stained) (% stained) (%) (%) (%) # type M C N M C N Cyto Cyto Cyto 1  6 0 80 80 0 0 30 70 40 80 2 31 0 0 90 0 40 0 3 52 0 25 50 0 0 70 0 20 20 4 16 0 0 40 0 0 30 0 5 0 5 58 0 0 50 0 0 10 0 0 0 6 52 0 80 70 0 0 50 0 5 0 7 53 0 0 80 0 0 30 0 10 10 8 52 0 50 90 0 0 20 60 10 20 9 31 0 80 80 0 0 50 70 20 40 10 16 0 50 80 0 0 50 60 20 10 11 no DNA 0 0 50 0 0 70 0 0 10 12 33 0 60 60 0 0 50 0 10 30 13 no DNA 0 70 80 0 0 70 0 20 10 14 52 0 0 70 0 0 70 0 30 20 15 no DNA 0 0 70 0 0 50 0 20 5 16 52 0 0 10 0 0 30 0 0 5 17 52 0 0 60 0 0 80 0 0 5 18 16 0 50 60 0 0 30 50 10 20 19 16 0 50 70 0 10 20 20 52, 44 0 50 80 0 0 40 0 30 30 21 16 0 0 50 0 0 50 0 20 20 22 16, 18, 6 0 0 40 0 0 0 0 10 0 23 16, 31 0 0 30 0 0 60 0 0 24  6 0 0 80 0 0 50 0 10 5 25 16 0 0 10 0 0 60 0 0 0 26 58 0 0 40 0 0 40 0 10 5 27 16, 39, 52 0 0 70 0 28  6 0 0 50 0 0 70 0 10 5 29 16 0 0 70 0 0 5 0 10 20 30 66, 68, 0 0 30 0 0 60 0 10 0

As shown in Table 3, nucleus staining are found in the dysplasia cells of all the CIN2 samples tested while only certain proportion of cases found staining of cytoplasm by the anti-E6 or anti-E7 antibody. The results indicate there is more staining of nucleus than cytoplasm of dysplasia cells found in CIN2 samples. As shown previously in SCC, ADC, and CIN3, HPV E7 protein in its adjacent normal epithelium cells was only found in nucleus, but not found in the cytoplasm of the epithelial cells. The staining of cytoplasm in CIN2 using anti-E6 antibody appears most distinguishable in dysplasia cells compared to its corresponding normal adjacent cells. The localization of the E6 proteins expressed in the cytoplasm of dysplasia cells, but not in the normal epithelium or stroma cells appears HSIL specific. These data demonstrate expression of HPV E6 proteins can be detected in the cytoplasm and nuclear of dysplasia cells of CIN2 tissues. For the cases with high level expression of HPV E6 proteins detected in the cytoplasm of dysplasia cells, it may suggest dysplasia progression. Similar staining pattern was also found when used other anti-HPV antibodies as shown in Table 3. The HPV IHC assay as described herein can be used to detect HPV early gene such as E6, E7, and late gene such as L1 proteins present in the dysplasia cells of CIN2.

Comparing the results of HPV IHC to the HPV DNA typing, the anti-E7 antibody reacts positively with all the HPV types present in the samples tested. For example, the anti-E7 monoclonal antibody as described herein can detect single HPV infection by at least, HPV-16, HPV-18, HPV-31, HPV-52, HPV-58, etc., which are cancer-related HPV types (high risk HPV types) and HPV6, HPV 53 which are not high-risk HPV types. The single anti-E7 monoclonal antibody can also detect HPV infection by two or more HPV types, such as the combination of HPV6, HPV-16, HPV-18, HPV-31, HPV-39, HPV-44, HPV-52, HPV-58, HPV-66, HPV-68, etc., which include most common high-risk HPV as well as low risk HPV types. These data indicate that the anti-E7 antibody described in this invention is non-type specific, able to detect HPV E7 proteins from common high-risk HPV types as well as low risk types in the CIN2 tissues. It is possible that formation of dysplasia cells is resulted from expression of oncoproteins, rather than genotyping of HPV types. It explains regression may occur for those infection by high-risk types with no detection of oncoproteins in cytoplasm. Thus, the HPV IHC assay described herein provides additional clinical information, not only for detection of HPV infection, but also for indication of dysplasia progression.

Since the staining of cytoplasm is, only found in dysplasia cells distinguishable to its corresponding normal cells, cytoplasmic staining was used to demonstrate the assay performance in dysplasia progression. To compare the specific cytoplasm staining of dysplasia cells in different stages of CIN or cancer tissues, data from Table 1-3 were further analyzed to obtain the assay performance. Percentage of staining 10% or above from each subject is considered positive, otherwise is negative for the sample less than 10% stained. As shown in table 4, the positive rate of the assay increases with the severity of CIN shown 38%, 52% to 100% for CIN2, CIN3, and SCC or ADE respectively. Positive predictive value (PPV) and negative predictive value (NPV) were also shown in this table to demonstrate the assay specificity using percentage staining of cytoplasm.

TABLE 4 Summary of the IHC staining results using anti-E7 on various biopsy samples during cervical cancer development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC Cyto. plasmic positive Speci- Anti-E7 stain stain rate ficity CIN2 Positive 11 0  38% 100% 100% PPV Negative 18 28  61% NPV CIN3 Positive 14 0  52% 100% 100% PPV Negative 13 28  68% NPV SCC Positive 11 0 100% 100% 100% PPV Negative 0 11 100% NPV ADC positive 10 0 100% 100% 100% PPV negative 0 10 100% NPV

TABLE 5 Summary of the IHC staining results using another anti-E7 on various biopsy samples during cervical cancer development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC Another Cyto. plasmic positive Speci- anti-E7 stain stain rate ficity CIN2 Positive 21 16 72% 57% PPV negative 8 9 36% 53% NPV CIN3 Positive 13 4 48% 76% PPV negative 14 25 86% 64% NPV SCC Positive 8 5 67% 62% PPV negative 4 6 55% 60% NPV ADC Positive 10 6 83% 63% PPV negative 2 6 50% 75% NPV

To show the expression of HPV E7 oncoproteins can be detected by another anti-E7 monoclonal antibodies, same tissue microarray was tested and shown in Table 5 representing the assay performance of another E7 antibody with positive rate of 72%, 48%, 67%, 83% for CIN2, CIN3, SCC and adenocarcinoma respectively. Positive predictive value (PPV) and negative predicative value (NPV) also shown in this table indicates the assay specificity using this antibody is not as good.

To validate the expression of HPV E6 oncoproteins can be detected by anti-E6 monoclonal antibodies, same tissue microarray were tested and shown in Table 6 and Table 7, representing the assay performance of two anti-E6 antibodies. E6 proteins are expressed in the cytoplasm of dysplasia cells from CIN2, CIN3, as well as tumor cells from invasive cancer samples can be detected by the anti-E6 antibody described in this invention. The same trend shown positive rate of the anti-E7 IHC assay increased with the severity of CIN is also found in the assay using anti-E6 antibody. The two anti-E6 antibodies show the same trend of increasing positive rate of assay over severity of CIN although one anti-E6 may give better assay performance than the other one. It is possible that MAb1 recognize different epitope from MAb7 does, thus give different assay performance. However, both monoclonal antibodies give high positive predictive value (PPV) and high negative predictive value (NPV) as shown in the tables. The overall positive rate of IHC assay using anti-E7 antibody is higher than using anti-E6 antibody. It is possible that E7 proteins are expressed earlier to serve as a biomarker for early detection of cervical cancer.

TABLE 6 Summary of the IHC staining results using anti-E6 on various biopsy samples during cervical cancer development and tissue lesions. dysplasia Normal IHC or Tumor Cyto- posi- Cyto. plasmic tive Speci- Anti-E6 stain stain rate ficity CIN2 Positive 5 1 17% 83% PPV Negative 25 29 97% 54% NPV CIN3 Positive 17 7 57% 71% PPV Negative 13 23 77% 64% NPV SCC Positive 7 1 64% 88% PPV Negative 4 10 91% 71% NPV ADC Positive 9 0 75% 100%  PPV Negative 3 12 100%  80% NPV total Positive 38 9 46% 0 81% PPV Negative 45 74 0 89% 62% NPV

To detect the expression of L1 viral proteins present in different stage of CIN, the same tissues microarrays were also tested on IHC assay using anti-L1 antibody. IHC score from L1 staining of cytoplasm was also obtained to analyze its positivity rate on all the samples. To study the correlation of HPV early and late proteins expressed in situ in different stage of CIN, Table 8 shows the positive rate of IHC assay with E7 expression and L1 expression in the cytoplasm for CIN2, CIN3, and invasive cancers. Since E7 seems a good biomarker for early detection of cervical caner, we compare the correlation of E7 and L1 expression in various stages of CIN and invasive cancer tissues using the HPV IHC assay described in this invention. For L1 IHC positive of CIN samples, as data shown in Table 8, about 60% (9 out of 15) of CIN2, or 58% (11 out of 19) of CIN3 show positive on E7 IHC assay. For L1 cytoplasmic positive of invasive cancer samples, 100% of both SCC (11 out of 11) and adenocarcinoma (10 out of 10) are E7 cytoplasmic positive, indicating 100% correlation of E7 expression with cancer progression. Data indicate that both L1 and E7 cytoplasmic positive of CIN2/3 may have higher risk in further dysplasia progression compared to those L1 positive but E7 negative on IHC assay.

TABLE 7 Summary of the IHC staining results using another anti-E6 on various biopsy samples during cervical cancer development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC Another Cyto. plasmic positive Speci- anti-E6 stain stain rate ficity CIN2 positive 5 0 17% 100% PPV negative 24 28 100%  54% NPV CIN3 positive 8 0 30% 100% PPV negative 19 29 100%  60% NPV SCC positive 11 0 92% 100% PPV negative 1 11 100%  92% NPV ADC positive 9 0 75% 100% PPV negative 3 12 100%  80% NPV total positive 33 0 41% 0 100% PPV negative 47 80 0 100%  63% NPV

TABLE 8 Summary of the IHC staining results using Anti-E7 compared with Anti-L1 and Anti-E6 on various biopsy samples during cervical cancer development and tissue lesions. CIN2 CIN3 SCC ADC Total No. of samples L1 (+) L1 (−) L1 (+) L1 (−) L1 (+) L1 (−) L1 (+) L1 (−) E7 positive 9 2 11  3 11  0 10  0 E7 negative 6 8 8 5 0 0 0 0 No. of samples E6 (+) E6 (−) E6 (+) E6 (−) E6 (+) E6 (−) E6 (+) E6 (−) E7 positive 5 6 8 6 11  0 9 1 E7 negative 0 17  0 13  0 0 0 0

Table 8 also shows the correlation of sample positivity with E7 expression and E6 expression in the cytoplasm. As data shown, for E7 cytoplasmic positive samples, about 45% (5 out of 11) of CIN2, or 57% (8 out of 14) of CIN3 show positive on E6 cytoplasmic expression, while 100% (11 out of 11) of SCC or 90% (9 out of 10) of ADE show E6 cytoplasmic expression. These data indicate that E6 may be expressed behind E7 during early dysplasia, but co-expressed in the late stage of cervical cancer.

In order to have homogeneous assay conditions for all stages of samples in one reaction, additional tissue microarray was generated to spot many samples from CIN2, CIN3, and invasive cancers on the same slide. Two tissue microarrays were prepared: One contains 10 individuals each for CIN2, CIN3, and SCC and their peripheral normal epithelia. One contains 10 individuals each for CIN2, CIN3, and ADE and their peripheral normal epithelia. To confirm the HPV IHC assay is specific to HPV related dysplasia or cervical cancer, additional tissue microarray containing more than 90 samples from various normal human tissues were also tested to use as the negative control of the HPV IHC assay. The same HPV IHC assays using anti-E6 and anti-E7 antibody were applied on these tissue microarray to obtain IHC staining percentage as described above. IHC staining of 10% or above shown in nucleus and/or cytoplasm was scored as positive of the assay.

All together, the IHC data from all the tissue microarrays tested herein were shown in Table 9-14. Table 9 shows IHC staining results using a mouse anti-HPVE7 antibody on various biopsy samples during cervical cancer development and tissue lesions. Data in Table 9 indicate that the presence of HPV E7 proteins in situ can be detected from various stages of cervical tissues with increasing positivity rate of assay from, CIN2, CIN3 to invasive cancer tissue like squamous cell carcinoma (SCC) or adenocarcinoma (AD). There is about 72%, and 90% positive rate for samples with CIN2 and CIN3 respectively. For cancer tissues (SCC and AD), 100% of samples stains positively by IHC using anti-HPV E7 antibody, indicating 100% of cancers expressing HPV oncogenic proteins. These data indicate the IHC assay using the HPV E7 antibody described in this invention provides a powerful tool to confirm the diagnosis of cervical cancer from the tissues in various grade.

TABLE 9 IHC staining results using a monoclonal anti-HPVE7 antibody on various biopsy samples during cervical cancer development and tissue lesions. IHC CIN2 CIN3 SCC ADC total Anti-HPV E7 34 43 22 23 122 positive Anti-HPV E7 13  5  0  0  18 negative Positive rate 72% 90% 100% 100% 87%

To obtain assay performance, data from table 9 were further analyzed. Table 10 shows summary of the IHC staining results from Table 9 using normal human tissues as CIN2 negative samples. Data indicate that the IHC staining method using the anti-HPV E7 antibody provides IHC assay sensitivity of 87% for CIN2+ with specificity of 92%. These data suggest this assay can be useful to detect HPV proteins for confirming of cervical lesion CIN2 or above.

TABLE 10 Summary of the immunohistochemistry staining results using a mouse monoclonal anti-HPVE7 antibody on CIN2+ lesions compared to CIN negative samples. CIN2+ CIN negative HPVE7 positive 122  7 95% PPV HPVE7 negative  18 85 83% NPV Sensitivity 87% Specificity 92%

To further analyze the data, Table 11 shows summary of the immunohistochemistry staining results from Table 9 and Table 10 indicating that using the anti-HPV E7 antibody described in this invention provides immunohistochemistry assay for CIN3+ (including CIN3, and invasive cancer) with 95% sensitivity, 92% specificity and 93% of positive predictive value, and 95% of negative predictive value. These data suggest this assay can be useful for clinical application to detect HPV proteins confirming cervical lesion in different stages.

TABLE 11 Summary of the immunohistochemistry staining results using a mouse monoclonal anti-HPVE7 antibody on CIN3+ lesions compared to CIN negative samples. CIN3+ CIN negative HPVE7 positive 88  7 93% PPV HPVE7 negative  5 85 94% NPV sensitivity 95% Specificity 92%

To confirm if expression of E6 protein can be detected by immunohistochemistry assay using the anti-E6 antibody described in this invention, the same tissues microarrays were performed on IHC assay using an anti-E6 antibody. As an example of another HPV immunohistochemistry assay, Table 12-14 show results of immunohistochemistry staining using anti-HPV E6 antibody. As data shown, HPV anti-E6 gives comparable immunohistochemistry results as HPV anti-E7.

TABLE 12 Immunohistochemistry staining results using a monoclonal anti-HPVE6 antibody on various biopsy samples during cervical cancer development and tissue lesions. IHC CIN2 CIN3 SCC AD total HPVE6 positive 32 35 23 17 107 HPVE6 negative 17 15  1  7  40 sensitivity 65% 70% 96% 71% 73%

Table 12 shows immunohistochemistry staining results using a mouse anti-HPVE6 antibody on various biopsy samples during cervical cancer development and tissue lesions. Data in Table 12 indicate that HPV E6 protein can be detected by the mouse monoclonal anti-HPV E6 antibody used in the immunohistochemistry assay described in this invention. The presence of HPV E6 proteins in situ can be detected from various stages of cervical tissues. As data shown, HPV E6 proteins are present in the samples with increasing positivity rate from, CIN2, CIN3 to cancer tissue like squamous cell carcinoma (SCC) or adenocarcinoma (AD). There is about 65%, and 70% positive rate for samples with CIN2 and CIN3 respectively. For cancer tissues, 96% of SCC samples stain positively by immunohistochemistry using anti-HPV E6 antibody, while only 71% of AD samples stain positively, indicating HPV E6 oncoproteins expressing more predominantly in SCC than in AD. These data indicate the immunohistochemistry assay using the HPV E6 antibody described in this invention provides a tool to confirm the diagnosis of cervical cancer from the tissues in various grade.

TABLE 13 Summary of the immunohistochemistry staining results using a mouse monoclonal anti-HPVE6 antibody on CIN2+ lesions compared to CIN negative samples. CIN2+ CIN negative HPVE6 positive 107 12 90% PPV HPVE6 negative  40 80 67% NPV Sensitivity 73% Specificity 87%

To further analyze the data, Table 13 shows summary of the IHC staining results from Table 11 and Table 12. As data indicated, using the anti-HPV E6 antibody provides immunohistochemistry assay sensitivity of 73% for. CIN2+ with specificity of 87%. These data suggest this assay can be useful for clinical application to detect HPV proteins confirming cervical lesion in different stages.

TABLE 14 Summary of the immunohistochemistry staining results using a mouse monoclonal anti-HPVE7 antibody on CIN3+ lesions compared to CIN negative samples. CIN3+ CIN negative HPVE6 positive 75  7 91% PPV HPVE6 negative 23 85 79% NPV sensitivity 77% Specificity 92%

To further analyze the data, Table 14 shows summary of the immunohistochemistry staining results from Table 13 indicating the immunohistochemistry staining method using the anti-HPV E6 antibody described in this invention provides immunohistochemistry assay for CIN3 or above with sensitivity of 77% specificity of 92% and 91% PPV, and 79% NPV. These data suggest this assay can be useful for clinical application to detect HPV proteins confirming cervical lesion in different stages.

The one or more immunological assays using antibodies and purified recombinant proteins derived from HPV early and/or late genes as obtained herein serve as reliable indicators whether HPV infection has occurred. In addition, HPV associated malignancy or pre-malignant cell transformation can be assayed. One of the most useful aspects of the invention is in diagnosing cervical carcinoma, both squamous cell and adenocarcinoma as well as any epithelial cell abnormality associated with oncogenic HPV infection including koilocytosis; hyperkerotosis; precancerous conditions encompasssing intraepithelial neoplasias or intraepithelial lesion; high-grade dysplasias; and invasive or malignant cancers.

In one embodiment, the early gene that can be used herein may include papillomavirus E6 genes, papillomavirus E7 genes, among others. In another embodiment, the late gene that can be used herein may include papillomavirus L1 genes, papillomavirus L2 genes, among others.

One aspect of the invention provides recombinant proteins, such as a recombinant hybrid protein containing a full length sequence of HPV oncogenic proteins, e.g., full-length E6, E7, and/or L1 polypeptide sequence, which have been found very difficult to obtain and purify due to undesirable aggregation during protein purification, protein instability, low levels of expression, low immunogenic responses of purified proteins. For example, many early E6 oncoproteins contain many cysteine amino acids and thus the correct topography of the E6 oncoproteins requires formation of many disulfide bonds properly. In addition, it was known that certain immunological assays using small peptides of early E6 and E7 proteins results in extremely low assay specificity and sensitivity and thus unsuitable as commercialized diagnostic tools.

In high grade CIN lesions, E6 and E7 are strongly expressed in host basal epithelial cells and interfere substantially with cell cycle control of these replication competent host cells. Expression of HPV oncoproteins interfers with G1-S-Phase regulation in host cells. The HPV E6 and E7 proteins target a plethora of cellular interactions, such as the inactivation of pRB by E7 and the degradation of p53 by E6. High level of HPV E7 proteins inactivates pRB and leads to disruption of E2F-Rb binding. Usually, binding of pRB to E2F blocks E2F driven cell cycle activation. In replicating cells, E2F is regulated by phosphorylation of RB. Rb phosphorylation is normally mediated by cyclin dependent kinases (CDK4, CDK6) that are controlled by several kinase inhibitors (INKs).

As a result of the loss of Rb/E2F repression and the strong activation by free E2F, the expression of a host cell protein, p16INK4a, is strongly overexpressed. In addition, S-phase genes are continuously activated since the p16INK4a mediated repression of Cdk4/6 has no downstream effect on pRb host cell protein. Since E7-dependent E2F release is not mediated by phosphorylation of pRb, the counter-regulatory p16INK4a expression has no effect on the activated cell cycle. Under physiological conditions p16INK4a is expressed when cells undergo a genomic stress situation such as substantial shortening of telomeres in ageing tissues. Also, apoptosis is abrogated by HPV E6 mediated degradation of p53. The overexpression of the cyclin dependent kinase (CDK) inhibitor, p16INK4a, is a direct consequence of deregulated HPV oncogene expression.

In addition, host cell proteins important for proliferation and host cell genome replication may be overexpressed as a result of HPV infection. These host cell proteins include, ki67 (MIB-1), MYC cellular oncogene, Cyclin proteins (e.g., cyclin A, B, E, etc.), CDKN2A/p16INK4a, telomerase (e.g., TERC), replication complex proteins (e.g., MCM5, CDC6, topoisomerase II alpha (TOP2A), MCM2, minchromosome maintenance proteins 2, 4, and 5, etc.).

As an example, the immunological assays for detection of HPV proteins, such as E6, E7, L1, etc., or immune response thereof due to HPV infection can be performed in high throughput ELISA screening assays, rapid immunological screening assays, and additional multiplexed protein chip assays, etc., and combinations thereof. Embodiments of the invention provides one or more assays, including an antibody, antigen, or immunocomplex assays developed to detect HPV viral proteins encoded by early genes (e.g., E6 and E7) and late genes (e.g., L1). In addition, the developed antibody, antigen, or immunocomplex assays for E6, E7, L1, protein or their antibodies thereof in one format, for example, a microplate format, can be adapted into a one-step immunochromatographic assay for the direct measurement of E6, E7, L1 proteins or antibodies induced by HPV infection.

The one or more protein chip assays, immunological assays, nucleic acid assays, as provided herein aims to employ user friendly procedures with simple instrument or no additional instrument to perform in a short period of time. Comparison of the results of the various immunological assays, nucleic acid hybridization assays with cytological and histological data for the human subjects as well as demographic information serve to validate the correlation and accuracy in diagnosing HPV infection and/or cervical cancer.

Another example of a method of screening a human subject infected with a human papillomavirus may include obtaining a clinical sample from the human subject, conducting a nucleic acid hybridization assay on the clinical sample, detecting the presence of a papillomavirus genome in the clinical sample from the human subject, conducting one or more immunological assays on the clinical sample, detecting the presence of an antibody to an early papillomavirus viral protein or the presence of the early papillomavirus viral protein in the clinical sample using a first recombinant protein of the early papillomavirus viral protein, and detecting the presence of an antibody to a late papillomavirus viral protein or the presence of the papillomavirus late viral protein in the clinical sample using a second recombinant protein of the late papillomavirus viral protein.

The one or more diagnostic immunological assays as described therein may also include obtaining polyclonal antibodies, monoclonal antibodies, and/or antiserum specific against the one or more recombinant proteins as obtained and described herein, taking a clinical sample likely to contain HPV associated proteins and/or antigens, reacting it with the obtained polyclonal antibodies, monoclonal antibodies, and/or antiserum specific for the one or more recombinant proteins, and assaying for the presence of any antibody-antigen complexes by suitable detection systems. Suitable detection system may employ various colormetric, chemilumenescent, flourescent substrates, etc., specific for a secondary antibody used in each immunological assay.

Early diagnosis of HPV infection is important for successful prevention and treatment of cervical cancer. Strategies to prevent cervical cancer requires improved HPV testing/screening to cover a broad range of the worldwide population in addition to closely follow-up those subjects with past or present HPV infection and/or pre-cancerous lesions. Importantly, it is known that infection in women for 12-15 years with HPV is required before invasive cancer to develop. It is thus important to be able to assay biomarkers for HPV infection as described herein to pre-screen women early, such that it will be possible to treat HPV infection early and prevent cervical cancer development, rather than having to rely on chemotherapy or radiation to treat cancer malignancy. 

1. A method of detecting papillomavirus infection in a human subject, comprising: conducting an immunological assay on a clinical sample of the human subject; and staining of a nuclear portion of a human cell from the clinical sample using one or more monoclonal antibodies capable of binding to HPV early viral proteins.
 2. The method of claim 1, wherein positive staining of the nuclear portion correlates with the progression to a disease stage selected from the group consisting of an early dysplasia stage, low-grade squamous intracervical lesion (LSIL), high-grade squamous intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2, CIN3) and combinations thereof.
 3. The method of claim 1, wherein positive staining of a cytoplasmic portion of the human cell indicates papillomavirus infection being progressed into a disease stage selected form the group consisting of a late dysplasia stage, cervical intraneoplasm (CIN3), invasive cervical cancer, squamous cell carcinoma (SCC), adenocarcinoma, and combinations thereof.
 4. The method of claim 1, wherein the one or more monoclonal antibodies are generated against one or more purified recombinant papillomavirus proteins.
 5. The method of claim 1, wherein the one or more monoclonal antibodies are also capable of binding to the HPV late viral proteins.
 6. The method of claim 1, wherein the one or more monoclonal antibodies further comprises an antibody capable of binding to the HPV late viral proteins.
 7. A method of detecting papillomavirus infection in a human subject, comprising: conducting an immunohistochemistry assay on humans cells from a clinical sample of the human subject using one or more antibodies generated against one or more purified recombinant papillomavirus proteins; and comparing the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cell from the clinical sample.
 8. The method of claim 7, wherein the one or more monoclonal antibodies are selected from the group consisting of anti-HPV E6 monoclonal antibodies, anti-HPV E7 monoclonal antibodies, anti-HPV L1 monoclonal antibodies, and combinations thereof.
 9. The method of claim 7, wherein more positive staining being observed in the nuclear portion than the cytoplasmic portion of the human cell from the clinical sample indicates papillomavirus infection in the human subject at an early disease stage selected form the group consisting of an early dysplasia stage, low-grade squamous intracervical lesion (LSIL), high-grade squamous intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2, CIN3), and combinations thereof.
 10. The method of claim 7 wherein more positive staining being observed in cytoplasmic portion than nuclear portion of the human cell of the clinical sample indicates papillomavirus infection in the human subject at a late disease stage selected from the group consisting of a late dysplasia stage, CIN3, invasive cancer stage, and combinations thereof.
 11. The method of claim 7, further comprising performing a hematoxylin and eosin stain on the clinical sample and comparing the results of the hematoxylin and eosin stain with the results of the one or more immunohistochemistry assays.
 12. The method of claim 7, further comprising performing a cytological papanicolaou smear assay on the clinical sample and comparing the results of the cytological papanicolaou smear assay with the results of the one or more immunohistochemistry assays.
 13. A method of detecting papillomavirus infection in a human subject, comprising: conducting an immunohistochemistry assay on a clinical sample of the human subject using two or more antibodies capable of binding to a HPV viral protein selected form the group consisting of E6, E7, L1 proteins, and combinations thereof; and comparing the staining of the human cell by the two or more antibodies, wherein positive staining of the human cell by at least one of the two or more antibodies indicates papillomavirus infection in the human subject.
 14. The method of claim 13, further comprising comparing the staining of the nuclear portion of the human cell from the clinical sample with the staining of the cytoplasmic portion, wherein positive staining of the cytoplasmic portion of the epithelial tissue sample indicates dysplasia progression by HPV infection.
 15. The method of claim 13, wherein the staining of the human cell from the clinical sample is compared using the two or more antibodies including an anti-HPV E6 antibody and an anti-HPV E7 antibody.
 16. The method of claim 13, wherein the staining of the human cell from the clinical sample is compared using the two or more antibodies including an antibody capable of binding to an early HPV viral protein and an antibody capable of binding to a late HPV viral protein.
 17. The method of claim 13, wherein the immunohistochemistry assay is used to detect HPV infection at various disease stages, the disease stage is selected from the group consisting of early stage HPV infection, late stage HPV infection, early stage cervical cell lesion, late stage cervical cell lesion, low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), cervical intraneoplasm stage 1, (CIN1), cervical intraneoplasm stage 2 (CIN2), cervical intraneoplasm stage 3 (CIN3), developed cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and combinations thereof.
 18. A kit for detecting papillomavirus infection in a human subject, comprising: an anti-HPV monoclonal antibody for performing an immunological assays on a clinical sample of the human subject, capable of staining a nuclear portion of one or more human cells from the clinical sample to compare the staining of the nuclear portion with the staining of the cytoplasmic portion of the human cells.
 19. The kit of claim 18, wherein the anti-HPV antibody is selected from the group consisting of an anti-HPV E7 monoclonal antibody, an anti-HPV E6 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E7 monoclonal antibody, a combination of an anti-HPV L1 antibody and anti-HPV E6 monoclonal antibody, a combination of an anti-HPV E6 antibody and anti-HPV E7 monoclonal antibody, and combinations thereof.
 20. The kit of claim 18, wherein the immunological assay is selected from the group consisting of ELISA (enzyme linked immunoabsorbant assays), antigen assays for papillomavirus proteins, antibody assays for antibodies against papillomavirus proteins, assays for papillomavirus immunocomplexes, protein chip assays, radioimmunoprecipitation assays, rapid membrane immunochromatographic assays, rapid stick immunochromatographic assays, immunohistochemistry for tissues and/or cervical cells, and immunocytological assays followed by flow cytometry, and combinations thereof.
 21. The kit of claim 18, wherein the anti-HPV monoclonal antibody is used to detect HPV infection at various disease stages, the disease stage is selected from the group consisting of early stage HPV infection, late stage HPV infection, early stage cervical cell lesion, late stage cervical cell lesion, low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL), atypical squamous cells of undetermined significance (ASCUS), cervical intraneoplasm stage 1, (CIN1), cervical intraneoplasm stage 2 (CIN2), cervical intraneoplasm stage 3 (CIN3), developed cervical cancer, adenocarcinoma (ADC), squamous cell carcinoma (SCC), and combinations thereof. 